Process for the preparation of retro-inverso peptides and 1,3-dioxane-4,6-dione intermediates thereof

ABSTRACT

A method of synthesis of a partially retro-inverso peptide incorporating at least one malonyl residue of formula (III) ##STR1## wherein R represents the side chain of an α-amino acid, is described which is characterized in that said malonyl residue is incorporated by condensing a 5-substituted-2,2-dimethyl-1,3-dioxane-4,6-dione of formula (VI) ##STR2## wherein R&#39; is the side-chain R wherein the functional groups, if any, are suitably protected, with an amino acid, amino acid amide, peptide fragment, or pseudo-peptide fagment wherein the terminal carboxyl group, if present, and the possible side-chain functionalities are suitably protected and the reactive amino group is tri-alkyl-silylated. 
     The new compounds of formula (VI) and a process for the preparation of a partially retro-inverso tuftsin analog which involves use of said method and said intermediate are also described and claimed.

The present invention relates to a new method of preparation of partialretro-inverso peptides and to the new intermediates used in such method.

"Retro-inverso" peptides are structural isomers of known peptides whichdiffer therefrom in the direction of one or more amide linkagescontained in the peptide sequence, which, in retro-inverso peptides, isreverted.

A "retro-inverso" peptide therefore will incorporate at least onegrouping of formula (I) ##STR3## wherein R and R₁ represent the sidechains of the suitable amino acids, and A represents nil or a peptidesequence containing at least one amino acid.

Reversal of said backbone peptide bond will be indicated, according tothe internationally recognized terminology, as

    --(g)AAA.sub.1 --A--(m)AAA--

wherein A has the same meaning as above, AAA and AAA₁ represent thestandard three letter codes which identify the amino acids having aradical R and R₁, respectively, in the side chain, the prefix (g),immediately before the designation for the amino acid residue AAA₁,indicates that said amino acid residue has been replaced by thecorresponding gem-diamino-alkyl derivative of formula (II) ##STR4##while the prefix (m) indicates that the amino acid residue AAA has beenreplaced by the corresponding malonyl derivative of formula (III)##STR5##

Reversal of one or more amide linkages generally stabilizes said bondsand the retro-inverso peptide which is thus obtained becomes moreresistant toward enzymatic degradation.

If, in case of biologically active peptides, the interaction withbiological receptors, which give rise to the particular activity, isessentially due to the side-chains, the retro-inverso analog willpresent the same pattern of biological activities of the so-called"parent peptide". Biological activity similar to the parent peptidecoupled with a higher stability, will render the new compound ofremarkable therapeutical interest.

For this reason, the retro-inversion approach has received a great dealof attention in the last few years and has been applied in a number ofcases with a fair rate of success (see for instance the partialretro-inverso LH-RH analogs described in Int.J.Peptide Protein Res. 17,1981, pp. 72-88; the partial retro-inverso enkephalinamides described inScience, 204, June 1979, pp.1210-12; the retro-inverso analogs ofSubstance P described in EP-A-161,007; the neurotensin analogs describedin U.S. Pat. No.-A-4,716,149; BPP_(5a) analogs described in EP-A-185433and EP-A-190597; equine angiotensinogen 5-14 decapeptide fragmentanalogs described in EP-A-127,235 and EP-A-127,234; tuftsin analogsdescribed in EP-A-253,190 and thymopentin analogs described inEP-A-282,891).

In general, the synthesis of retro-inverso peptides is carried out withmethods which are substantially analogous to those conventionallyemployed for the preparation of peptides and which involve sequentialcondensation of the different residues (or fragments) present in thechain, differing therefrom for the introduction of at least one malonylresidue and at least one gem-diaminoalkyl residue.

Introduction of the gem-diaminoalkyl residue of formula (II) can beeasily achieved by using, in the formation of the peptide bond with afree carboxyl group, the corresponding amide of formula (IV) ##STR6##wherein R₁ ' represents the side-chain R₁ wherein the functional groups,if any, are suitably protected, and then converting the terminal amidogroup in primary amino group by reaction with TIB (see Italian PatentApplication no. 221281 A/81).

Introduction of a malonyl residue is actually carried out using2-substituted malonic acid mono-esters of general formula (V) ##STR7##wherein R" is a lower alkyl group and R' represents the side chain Rwherein the functional groups, if any, are suitably protected.

Said mono-esters (V) are obtained by monosaponification of thecorresponding diesters which are prepared by conventional chemicalprocesses for the preparation of 2-substituted malonates.

However, depending on the meaning of R', preparation of large amounts ofmono-esters of high purity, as typically required in sequentialprocesses, may be very difficult.

In general, in fact, preparation of the diester leads to a mixture ofproducts from which the desired diester cannot be easily recoveredunless chromatographic purification techniques are employed.Furthermore, owing to the poor stability of the mono-ester, itspreparation by monosaponification of the diester, must be considered aspart of the multistep process needed to incorporate it in the peptidesequence, process which involves, besides said monosaponification,activation, coupling and final saponification steps.

As an example, preparation of2-(4-tert-butoxycarbonyl-amino-butyl)malonic acid mono ethyl ester,[(m)Lys(Boc)OEt], starting from 4-amino-1-butanol (a multi-step processwhich also includes chromatographic purification of the di-ester),occurs with overall yields lower than 20% (see P. V. Pallai et al.,Biochemistry, 24, (1985), 1933-41).

Said procedure has the additional disadvantage of drastically limitingthe choice of protecting groups in the overall strategy of synthesis, asthese groups must be stable in the conditions used for the selectiveremoval of the ester group from the incorporated malonyl residue.

Alternatively, the use of Meldrum's acid derivatives has been proposed afew years ago (see M. Goodman and M. Chorev in "Perspectives in PeptideChemistry"- Ed. Eberle, Geyer & Wieland- (1981) - P. 283 et seq.), and,more particularly, the use of a5-substituted-2,2-di-methyl-1,3-dioxane-4,6-dione of formula (VI)##STR8## wherein R' represents the side-chain R wherein the functionalgroups, if any, are suitably protected.

In the actual practice, the efforts made to introduce a (m)Ala residue,using 2,2,5-trimethyl-1,3-dioxane-4,6-dione (the compound of formula(VI) wherein R' is methyl--Meldrum's acid 5-substituted with alanineside-chain--shortly indicated as (M)Ala), gave unsatisfactory results.In fact, only partial N-acylation of the amino acid partner has beenobserved and the desired pseudo-dipeptide has been obtained with lowyields (see Italian patent application 23417 A/82). It has nowsurprisingly been found that Meldrum's acid derivatives of formula (VI)can satisfactorily be used in the preparation of retro-inverso peptides,to incorporate a malonyl residue (III), provided the condensationreaction "partner" is employed as the corresponding derivativetri-alkyl-silylated at the reactive amino nitrogen.

As a "partner" in the condensation reaction, an amino acid, an aminoacid amide, a peptide fragment, or a pseudo-peptide fragment to which,in the desired pseudo-peptide sequence, the malonyl group must be linkedand whose side-chain functionalities, if any, are suitably protected,can be employed.

It is apparent that, when a solid-phase condensation strategy isemployed, said "partner" may be attached via the C-terminus to asuitably selected insoluble support as known in this field (Merrifieldsolid-phase peptide synthesis); in particular, in the solid-phasesynthesis of retro-inverso peptides according to the most widely knownmethod (see A. Pessi et al., J.Chem.Soc., Chem. Commun. (1983), 195),the compounds of formula (VI) are conveniently employed for thepreparation of the necessary mono-malonyl amino acid amides.

For the purposes of the present invention, therefore, the term"N-tri-alkyl-silyl derivative" refers to the compound obtainable from anamino acid, an amino acid amide, a peptide fragment or a pseudo-peptidefragment, whose side-chain functional groups, if any, and whose terminalcarboxy group, if any, are suitably protected, through activation of theamino group with a tri-alkyl-silyl group; said starting amino acid,amino acid amide, peptide fragment or pseudo peptide fragment optionallybeing anchored to an insoluble support.

The term "alkyl" identifies a straight or branched alkyl chaincontaining from 1 to 6 carbon atoms, and, preferably, methyl or ethyl.

Condensation of a compound of formula (VI) with the suitably selectedN-trialkyl-silyl derivative, readily affords effective acylation of theamino group.

An additional advantage deriving from the use of this method toincorporate the malonyl residue, resides in the possibility, by simplehydrolysis of the obtained product at neutral or slightly acidic pH, ofsetting free the carboxyl group of the pseudo-peptide terminal malonylresidue, which is then available for the formation of the next amidebond.

In the actual practice, condensation between Meldrum's acid derivative(VI) and the suitably selected N-tri-alkyl-silyl derivative, is carriedout by contacting a compound of formula (VI) wherein R' is as definedabove, with at least an equimolar amount, and preferably a slightexcess, of the N-tri-alkyl-silylated reaction partner.

The two reactants are contacted in the presence of an inert organicsolvent. In general, polar, aprotic, organic solvents such as, forinstance, halogenated aliphatic or aromatic hydrocarbons, e.g. methylenechloride, dichloroethane, chloroform, chlorobenzene, etc., cyclic orlinear ethers, e.g. tetrahydrofuran, dioxane, diethyl ether, etc., andetherated glycols, e.g. ethylene glycol mono-methyl or mono-ethyl ether,can conveniently be employed.

Condensation is typically carried out at a temperature of from 0° C. tothe reflux temperature of the reaction mixture and, preferably, at atemperature of from 15° to 50° C.

When condensation is complete, a mild acidic hydrolysis, using a dilutedaqueous solution of an organic or inorganic acid, e.g. 5% or 10% citricacid, or 1% hydrochloric acid solution, affords the desiredpseudo-peptide sequence incorporating a terminal malonyl residue with afree carboxyl group.

Any other step in the overall synthesis of the desired retro-inversopeptides, either before or after the introduction of the malonylresidue, may be carried out according to techniques widely known inpeptide chemistry and conventionally employed in peptide synthesis.

The starting N-tri-alkyl-silylated derivative is conveniently preparedaccording to methods known in literature, using the conventionalsilylating agents.

Typically, said methods involve the use of tri-alkyl-silyl halides orN,O-bis-tri-alkyl-silylacetamides as the silylating agents.

In particular, as an example, the desired N-tri-alkyl-silyl derivativescan be prepared by reacting the suitably selected amino acid, amino acidamide, peptide or pseudo-peptide sequence, with at least an equimolaramount, but preferably an excess thereto, ofN,O-bis-tri-alkyl-silylacetamide, in the presence of a polar and aproticorganic solvent which does not negatively interfere with the reactioncourse, at a temperature generally of from room temperature to thereflux temperature of the reaction mixture.

At the end of the silylation reaction, the obtainedN-tri-alkyl-silylated derivative can be separated by conventionalmethods, e.g. by distillation or extraction.

Alternatively, and preferably, the condensation reaction is carried outdirectly adding the compound of formula (VI) to the mixture derivingfrom the silylation reaction.

In this case, to ensure that activation of the amino nitrogen proceedsquantitatively, silylation is preferably carried out using a strongexcess of silylating agent, e.g. 2-3 moles of silylating agent per moleof substrate. Said excess is also useful to neutralize traces ofhumidity in the reaction mixture.

It is also possible, and this represents a further preferred embodimentof the present invention, to prepare the N-trialkyl-silyl derivative,directly in situ, when carrying out the condensation reaction. In thiscase the condensation reaction is carried out by contacting the compoundof formula (VI) with the amino acid, amino acid amide, peptide orpseudo-peptide sequence with the free terminal amino group, and thesilylating agent. Also in this case, preferably, a strong excesssilylating agent is employed, while the reaction conditions are thosereported above.

Some of the starting compounds of formula (VI), in particular thosecompounds of formula (VI) wherein R' represents the side chain ofphenylalanine (R'=--CH₂ --C₆ H₅), alanine (R'=--CH₃), valine(R'=--CH(CH₃)₂), isoleucine (R'=--CH(CH₃)CH₂ CH₃), glutamic acid γ-ethylester (R'=--CH₂ CH₂ COOC₂ H₅) and tryptophan (R'=--CH₂ (3-indolyl)), areknown compounds which can be synthesized according to methods known inliterature.

In general, however, all the compounds of formula (VI) can be preparedby alkylation of the 5-positioned carbon atom of Meldrum's acid offormula (VII) ##STR9## with methods which differ depending on thestructure of the substituent R' which has to be introduced.

As an example, one of such methods is the reaction of Meldrum's acid(VII) with ketones or aromatic or α-branched aliphatic aldehydes, toafford Knoevenagel-like products which are then easily reduced withsodium borohydride to yield 5-alkyl substituted Meldrum's acids (A. D.Wright et al., Tetrahedron Lett., pp.1759-62).

Using linear aliphatic aldehydes, Knoevenagel-like products cannot beisolated because the process mainly affords bis-adducts deriving from asubsequent Michael reaction; in this case therefore a one-pot reductivealkylation of Meldrum's acid is carried out using a reducing agent whichselectively acts on double bonds (D. M. Hrubowchak et al., TetrahedronLett., (1979), pp. 2325-6).

An alternative method for synthetizing these compounds consists in theMichael addition of the Meldrum's acid anion to electrophilic olefins(Cheng-Chu Chan et al., Synthesis, (1984), pp. 224-5).

The procedures actually employed for the preparation of the compounds offormula (VI) are substantially those described in the above citedreferences with optional minor modifications, apparent to any chemist,which are made to fit the methods to the particular substrates.

As for the protecting groups which may be present on the 5-positionedsubstituent, the protecting groups of the side-chain functionalitiesknown in literature and conventionally employed in peptide syntheses canbe used. In some cases, there might be some limitations to theprotecting groups of the radicals which must be introduced at the5-position, due to the particular process employed for the preparationof the compound of formula (VI).

It is however always possible, once the 5-substituted Meldrum's acidderivative has been synthetized, to replace the protecting groupemployed in the preparation of this product with a different one, mostlysuitable for peptide synthesis.

The process of the present invention may be employed for the synthesisof any peptide containing at least one retro-inverso peptide bond. Inparticular, optimum results have been obtained in the synthesis of thetuftsin analog, described in EP-A-253,190, which contains aretro-inversion at the Thr-Lys bond.

It represents therefore a further object of the present invention aprocess for the synthesis of a peptide of formula

    (g)Thr--(R,S)(m)Lys--Pro--Arg--OH

characterized in that incorporation of the malonyl residue of formula##STR10## is carried out using a 5-substituted Meldrum's acid of formula(VIa) ##STR11## wherein P^(L) is a protecting group of lysine aminofunction.

As anticipated P^(L) is any of the conventional amino protecting groupsand, preferably, trifluoroacetyl, tert-butoxycarbonyl,tert-amyloxycarbonyl, or optionally nitro- or halo-substitutedbenzyloxycarbonyl.

The compounds of formula (VIa) are conveniently prepared starting from4-amino-butyraldehyde diethyl acetal. In particular, the compound offormula (VIa) wherein P^(L) is a trifluoroacetyl group has been preparedthrough N-acylation of 4-amino-butyraldehyde diethyl acetal withtrifluoroacetic anhydride, followed by hot hydrolysis of the obtained4-trifluoroacetamido-butyraldehyde diethyl acetal in slightly acidicaqueous solution. A mixture of products is obtained which may beemployed as such in the reductive alkylation of Meldrum's acid (VII)affording the compound of formula (VIa) wherein P^(L) is atrifluoroacetyl group.

If desired, alkaline hydrolysis of the trifluoroacetyl group followed byreaction with a different acylating agent can easily afford any othercompound (VIa).

According to a particularly preferred embodiment, the process forpreparing the tuftsin analog retro-inverted at the Thr-Lys bond involves:

a) condensing the compound of formula (VIa) with the Nα-tri-alkyl-silylderivative of (D)-threonineamide wherein the hydroxy group is suitablyprotected by a protecting group P^(T),

b) hydrolysing in mild acidic conditions the thus obtained product toafford a fragment of formula (VIII) ##STR12## wherein P^(T) and P^(L)are as defined above, c) condensing the fragment of formula (VIII) witha dipeptide fragment of formula (IX) ##STR13## wherein P^(C) is acarboxyl protecting group and P^(G) is an arginine guanidino protectinggroup, d) converting the terminal threonine amide group into an aminogroup by treatment with TIB and

e) removing all the protecting groups.

Other retro-inverso peptides which are conveniently prepared by themethod of the present invention, wherein the malonyl residue isincorporated by condensing the corresponding 5-substituted Meldrum'sacid derivative with the suitably selected N-tri-alkyl-silyl derivative,are desmorphin retro-inverso analog described in Chemical Abstracts 103178616, and thymopentin (TP5) retro-inverso analog described inEP-A-282,891. While in the latter case a Meldrum's acid derivative offormula (VIa) has been employed, in the former case the startingMeldrum's acid derivative has the following formula (VIb) ##STR14##wherein P^(O) is a protecting group of tyrosine hydroxy function. Alsoin this case any protecting group, conventionally employed in peptidesynthesis for the protection of tyrosine hydroxy group, can suitably beemployed. Particularly preferred groups are however tert-butyl,tert-amyl, benzyl, optionally halo- or nitro-substituted, andtriflucroacetyl.

A further object of the present invention are therefore the newcompounds of formula (VI), and in particular the new compounds offormula (VIa) and (VIb), useful as intermediates in the synthesis ofretro-inverso peptides.

The following examples which are only aimed at better illustrating somerepresentative embodiments of the present invention should not beinterpreted as a limitation to the scopes thereof.

EXAMPLE 1 [(g)Thr¹,(R,S)(m)Lys² ]tuftsin acetate 1) (M)Lys(TFA)(2,2-dimethyl-1,3-dioxane-5-(4-trifluoroacetamido-butyl)-4,6-dione)

Dimethylaminopyridine (25.6 g, 210 mmol) and, dropwise, a solution oftrifluoroacetic anhydride (29.4 ml, 210 mmol) in methylene chloride (100ml) are added to a solution of 4-aminobutyraldehyde diethyl acetal (32.2g, 200 mmol) in methylene chloride (600 ml) cooled to 0° C. and keptunder stirring. The mixture is stirred at 0° C. for additional 30minutes, the precipitate which forms is removed by filtration and thefiltrate is washed with water (4×200 ml). The organic phase is driedover Na₂ SO₄ and evaporated under vacuum affording an oily product (48g). 1N HCl (500 ml) is then added thereto under vigorous stirring, andstirring at room temperature is continued until a homogeneous solutionforms (about 15 minutes). The solution is then cooled and the pH isbrought to 6 by the addition of 1N NaOH. The solution is then heated to100° C. for 15 minutes, cooled and brought to a small volume (about 200ml) under vacuum. The solution is extracted several times with methylenechloride and the organic extracts, pooled, are dried over Na₂ SO₄ andevaporated under vacuum yielding an oily residue (27 g) containing 60%by weight of 4-trifluoroacetamido-butyr-aldehyde.

The obtained product is added to a solution of sodium cyanoborohydride(3.3 g, 52 mmol) and 2,2-dimethyl-1,3-dioxane-4,6-dione (Meldrum's acid)(12 g, 84 mmol) in N,N'-dimethylformamide (DMF) (50 ml).

After stirring at room temperature for 1 h, water (150 ml) is addedthereto and pH is brought to 4.5 affording a precipitate which isrecovered by filtration, washed with a small amount of cold water andthen with ethyl ether and finally dried under vacuum. The yield is 17.5g (28%), m.p. 131°-2° C.

Elemental analysis % Calculated for C₁₂ H₁₆ O₅ NF₃ :C 46.3; H 5.15; N4.5; % Found C 46.5; H 5.3; N 4.6;

Mass and ¹ H-NMR analyses confirm the assigned structure.

2) HO(m)Lys(TFA)-D-Thr(Bu^(t))NH₂(N,2-(4-trifluoroacetamido)butyl-malonyl-(O)-tert-butyl-(D)-threoninecarboxamide

N,O-bis-trimethylsilylacetamide (8.1 g, 40 mmol) is added to a solutionof O-tert-butyl-D-threoninecarboxamide (3.5 g, 20 mmol) in methylenechloride (90 ml) and the solution is refluxed under stirring for 6 h.After cooling the solution to room temperature, the product obtained instep 1) above (5.6 g, 18 mmol) is added thereto and the reaction mixtureis stirred for 18 h. The mixture is washed a few times with 5% citricacid aqueous solution, the solvent is evaporated off and the thusobtained residue is triturated with a water/acetone mixture. Anamorphous solid (5.2 g) is obtained whose identity has been confirmed by¹ H-NMR and mass (electronic impact) analyses.

3) [(m)Lys-D-Thr(Bu^(t))NH₂ ]-Pro-Arg-OH

The product obtained in the foregoing step (2.1 g. 5 mmol) is dissolvedin methylene chloride (30 ml)/DMF (4 ml), the obtained solution iscooled to 0° C. and N-hydroxybenzotriazole (HOBt) (0.67 g, 5 mmol) andN,N'-dicyclchexylcarbodiimide (DCC) (1 g, 5 mmol) are then stirred in.After stirring at 0° C. for 30 minutes and at room temperature foradditional 30 minutes, N,N'-dicyclohexylurea (DCU) that forms is removedby filtration and HCl·Pro-Arg(NO₂)-OBz (2.2 g, 5 mmol) (prepared asdescribed by M. Fridkin et al. in Biochim. Biophys. Acta, 496,pp.203-11, (1977)) and triethylamine (0.7 ml, 5 mmol) are added to thefiltrate.

After stirring at room temperature for 18 h, the filtrate solution isevaporated under vacuum, the residue is taken up in AcOEt (100 ml) andthe thus obtained solution is washed in succession with 5% NaHCO₃solution, 10% citric acid solution and water.

The organic phase is dried over Na₂ SO₄ and the solvent is evaporatedoff yielding a solid (3.8 g) which is then dissolved in methanol (50ml). Palladium sponge (4 g) and ammonium formate (0.95 g, 15 mmol) areadded to the thus obtained solution. The mixture is stirred gently for 2h at room temperature, the catalyst is then removed by decantation andwashed with methanol.

The methanol phases are combined and evaporated under vacuum to a volumeof about 20 ml. Water (30 ml) is then added thereto and pH is brought to12 by the addition of 1N NaOH. After 45 minutes at room temperature, thepH of the solution is adjusted at 5 by the addition of 1N HCl, thesolution is partially evaporated under vacuum and lyophilized from waterfew times.

The desired product is then obtained by reverse-phase displacementchromatography of the solid residue, using a Lichroprep® RP-18 column asthe stationary phase, 0.1% trifluoroacetic acid aqueous solution as thecarrier and 50 mM benzyl-tributyl-ammonium chloride as the displacer.Titer and purity of the components of the mixture in the differentfractions are evaluated by reverse-phase HPLC. Working on an aliquot ofthe raw solid (1.2 g), the pure compound (0.92 g) is obtained whoseidentity has been confirmed by ¹ H-NMR and mass (FAB) analyses.

4) (AcOH)₂ ·H-(g)Thr-(m)Lys-Pro-Arg-OH({(2)-2[N-(1-amino-2-hydroxy-propyl)carbamyl]-6-amino}hexanoyl-L-prolyl-argininediacetate)

The compound obtained in the preceding step (0.7 g, 1 mmol) is dissolvedin acetonitrile/water 1/1 (40 ml) andI,I-bis-trifluoroacetoxy-iodo-benzene (TIB) (0.56 g, 1.3 mmol) isstirred in under nitrogen atmosphere. After stirring for 6 h, themixture is evaporated under vacuum and the obtained residue is suspendedin concentrated HCl and kept at 0° C. for 8 minutes. The mixture is thenevaporated under vacuum a few times, taking up the residue in water, andfinally it is lyophilized. The thus obtained residue is then purified bychromatography on a CM-Sephadex C-25 (2.6×30 cm) column, eluting with alinear gradient of 0.05-0.5M ammonium acetate, flow rate 5 ml·min⁻¹ to atotal volume of 2 1.

Lyophilization of the pure fractions affords a solid (0.31 g) whosestructure has been confirmed by ¹ H-NMR and mass (FAB) analyses.

EXAMPLE 2 [(g)Arg¹, (R,S)(m)Lys² ]TP5 acetate 1) (M)Lys(Boc)(2,2-dimethyl-5-(4-tert-butoxycarbonylamino-butyl)-1,3-dioxane-4,6-dione)

A solution of the compound obtained in step 1) of Example 1-/(M)Lys(TFA)] (6.22 g, 20 mmol) in water (50 ml) is brought to pH 12.5by the addition of 2N NaOH. The mixture is stirred at room temperaturefor 30 minutes and then cooled to 0° C. A solution ofdi-tert-butyl-carbonate (8.72 g, 40 mmol) in dioxane (75 ml) is addedthereto and the reaction mixture is allowed to warm up to roomtemperature and stirred for 2 h keeping the pH at 9. Dioxane isevaporated off under reduced pressure and the aqueous solution is washedwith n-hexane. The pH is brought to 3.5 by the addition of citric acidand the solution is extracted few times with methylene chloride. Theorganic extracts are pooled, dried over Na and evaporated to afford awhite solid (5.4 g, 86%) with m.p. 112°-3° C.

¹ H-NMR confirms the assigned structure.

2) Ho(m)Lys(Boc)-Asp(Bu^(t))-Val-Tyr(Bu^(t))OBu^(t)

A solution of ammonium formate (1.3 g, 20 mmol) in methanol (20 ml) andpalladium sponge (3.5 g) are added under nitrogen atmosphere to asolution of Z-Asp(Bu^(t))-Val-Tyr(OBu^(t))-OBu^(t) (prepared asdescribed in Example 1 step 3) of EP-A-282,891) (4.94 g, 7 mmol) inmethanol (80 ml). The resulting solution is heated for a short period oftime to 40° C. and then kept at room temperature for 1 h. The mixture isallowed to settle, then it is filtered on celite, washing the solid onfilter with methanol. The solvent is evaporated off, and the obtainedresidue is taken up in AcOEt. The organic solution is washed with 10%Na₂ CO₃ solution, with water, and finally dried on MgSO₄.

Evaporation of the solvent gives the compoundH-Asp(Bu^(t))-Val-Tyr-(Bu^(t))OBu^(t) as an oily product (3.75 g, 6.66mmol).

Tri-methyl-chloro-silane (0.825 ml, 6.66 mmol) is added to a solution ofthe thus obtained product, the compound prepared according to step 1)above (2.3 g, 7.32 mmol), and N,O-bis-trimethylsilyl-acetamide (3.33 ml,13.32 mmol) in tetrahydrofuran (60 ml). The reaction mixture is allowedto stand at room temperature overnight, then the solvent is removed andthe residue is taken up in methylene chloride. The solution is washedwith aqueous citric acid pH 4, dried over MgSO₄ and brought to a smallvolume. Addition of AcOEt/n-hexane precipitates the desired product(4.35 g, 76%).

¹ H-NMR and FAB-MS analyses confirm the assigned structure.

3) [(m)Lys(Boc)-(D)-Arg(Mtr)NH₂ ]-Asp(Bu^(t))-Val-Tyr(Bu^(t))-OBu^(t)

The compound obtained in the foregoing step (1.23 g, 1.5 mmol) and HOBt(0.202 g, 1.5 mmol) are dissolved in methylene chloride (30 ml)containing DMF (1 ml) cooled to 0° C., and DCC (0.309 g, 1.5 mmol) isadded to the obtained solution. After 30 minutes at 0° C., the reactionmixture is brought to room temperature and stirred for additional 30minutes. DCU is then filtered off and N^(G)-(4-methoxy-2,3,6-trimethyl)benzenesolfonyl-D-arginine-amide (0.580 g,1.5 mmol) is added to the remaining solution. The reaction mixture isallowed to stand at room temperature overnight, then the solvent isremoved, the residue is taken up in AcOEt, the organic solution iswashed in succession with 5% NaHCO₃ solution, 10% citric acid solution,and water. The solution is dried over MgSO₄, the solvent is evaporatedoff and the residue is triturated with n-hexane, yielding the desiredproduct (1.63 g, 90%).

4) AcOH H-(g)Arg-(R,S)[m]Lys-Asp-Val-Tyr-OH

The compound of the foregoing step (1.187 g, 1 mmol) is dissolved in H₂O/CH₃ CN/DMF (35/60/5) (20 ml) and, while keeping the solution undernitrogen atmosphere, TIB (0.473 g, 1.1 mmol) is added thereto. Themixture is kept at room temperature overnight, then evaporated; theresidue is taken up in ethyl ether and the solution is evaporated again.This last procedure is repeated three times, then a mixture oftrifluoroacetic acid/trifluoromethanesulfonic acid/ethanedithiol(89/1/10) (60 ml) cooled to 0° C. is added thereto. After 10 minutes at0° C., triethylamine (0.9 ml) is added, the temperature is brought to40° C. and the mixture is evaporated under a nitrogen stream. Theresidue is taken up in water (100 ml) and washed with ethyl ether (100ml). The organic phase, in its turn, is washed with water (50 ml) andthe aqueous phases are combined, washed again with ethyl ether (3 x 50ml) and lyophilized. Purification of the thus obtained product by ionexchange chromatography is carried out as described in Example 1, step11 of EP-A-282,891.

EXAMPLE 3 Retro-inverso analog of desmorphinH-Tyr-D-Ala-(g)Phe-Gly-(m)Tyr-Pro-Ser-NH₂ 1) (M)Tyr(Bu^(t))(2,2-dimethyl-5-(4-tert-butoxy)benzyl-1,3-dioxane-4,6-dione)

N,N-dimethylformamide di-tert-butyl acetal (48 ml, 200 mmol) is added(0.1 ml/min) to a solution of 4-hydroxybenzaldehyde (6.1 g, 50 mmol) inbenzene (50 ml) kept under nitrogen atmosphere at 80° C. The mixture isthen cooled to room temperature and evaporated under reduced pressure.The residue is dissolved in methylene chloride, washed with 5% NaHCO₃solution and then with water, dried over Na₂ SO₄ and evaporated to adark oil. Said oil is purified on a silica gel column eluting withAcOEt/n-hexane 2/8 (v/v). The purified product (5 g, 28 mmol) is addedto a solution of Meldrum's acid (4.46 g, 31 mmol) and piperidine (0.6ml, 6 mmol) in DMF (30 ml). After stirring at room temperature for 3 h,the solvent is evaporated off under reduced pressure and the residue isdissolved in methanol (50 ml). Solid NaBH₄ (1.14 g, 30 mmol) isgradually added to the thus obtained solution within 15 minutes, thenthe reaction is stopped by the addition of water (100 ml) and 1N HCl upto pH 3. The resulting precipitate is recovered by filtration, washedwith cold water, dried under vacuum and triturated with hexane yielding6.12 g (40%) of (M)Tyr(Bu^(t)) (Meldrum's acid derivative of formula(VIb) wherein P^(O) is a tert-butyl group) with m.p. 75°-7° C.

The ¹ H-NMR analysis confirms the assigned structure.

The compound of the foregoing step is employed in the synthesis of thecompound of the title by following substantially the scheme reported inChemical Abstracts 103 178616p, with the difference that the (m)Tyrresidue is incorporated by condensing the N-trimethyl-silyl derivativeof the fragment H-Gly[Z-D-Ala-(g)Phe] with the compound obtained instep 1) above.

The condensation reaction is carried out substantially as described instep 2) of Example 2.

We claim:
 1. A 5-substituted-2,2-dimethyl-1,3-dioxane-4,6-dione offormula (VI) ##STR15## wherein R' represents --CH₂ --CH₂ --CH₂ --CH₂--NH₂, the side-chain of the amino acid lysine, or ##STR16## theside-chain of the amino acid tyrosine, wherein the functional groups aresuitably protected.
 2. A compound of claim 1 wherein R' is --CH₂ --CH₂--CH₂ --CH₂ --NH₂, the side-chain of the amino acid lysine, wherein theamino group is protected with a group selected from trifluoroacetyl,tert-butoxy-carbonyl, tert-amyloxycarbonyl, and, optionally nitro- orhalo-substituted, benzyloxy-carbonyl.
 3. A compound of claim 1 whereinR' represents ##STR17## the side-chain of the amino acid tyrosine,wherein the hydroxy group is protected with a group selected fromtert-butyl, tert-amyl, benzyl, halo- or nitro-substituted benzyl, andtrifluoroacetyl.